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						<h2><strong>BLH</strong><br /><span style="font-size:0.8em; text-transform:lowercase; font-weight:300">Endogenous production of retinal from &beta;-carotene </span></h2> 
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									<h3 class="wow fadeInDown"><span style="text-transform:lowercase">&beta;</span>-carotene to Retinal</h3>
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										<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/1/1f/Unitn_project_results_blh_retinal.png" title="Engineered biochemical pathway for the biosynthesis of retinal in E. coli"><img src="https://static.igem.org/mediawiki/2015/6/67/Unitn_project_results_blh_retinal_thumb.jpg" alt="" style="width:100%; max-width:965px;"/></a>
										<p class="image_caption"><span> Engineered biochemical pathway for the biosynthesis of retinal in <i> E. coli </i></span> The genes &beta;-carotene biosynthesis (ctrEIBY) and cleavage (blh) are engineered in <i> E. coli </i>. pharnesyl di phosphate, the starting molecule of the pathway is naturally built in <i> E. coli </i></p>
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										 <p>Proteorhodopsin needs all<i>trans</i>-retinal to be active. In many bacteria that naturally produce proteorhodopsin (i.e. SAR86), retinal is synthetized starting from &beta;-carotene. These bacteria have a cluster of genes that includes in addition to proteorhodopsin, the operon for &beta;-carotene production ctrEIBY and blh that encodes for &beta;-carotene 15,15’-Dioxygenase<sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_1')" href="#refs_1">[1]</a></sup>.</p>
								 
										<p>We built two different devices for the production of retinal and one for the production of &beta;-carotene. We used parts newly built by us and other extracted by the registry (kit 2012).</p>
										
										<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/1/10/Unitn_pics_project_blhdevice_corrected.png" title="Devices for the production of retinal"><img src="https://static.igem.org/mediawiki/2015/1/10/Unitn_pics_project_blhdevice_corrected.png" alt="" style="width:100%; max-width:965px;"/></a>
										<p class="image_caption"><span>Devices for the production of retinal</span> To provide blh and &beta;-carotene synthesis to E. coli, we designed the new part <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604020" target="_blank">BBa_K1604020</a> starting from <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_274210" target="_blank">BBa_274210</a>. We finally merged the two together <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604022" target="_blank">BBa_K1604022</a>.</p>
 
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									<h3 class="wow fadeInDown">Orange is the new black</h3>
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										<p>Our &beta;-carotene device was built with the arabinose promoter from UniTN 2012 (<a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K731201" target="_blank">BBa_K731201</a>) and the &beta;-carotene part of Cambridge 2009 extracted from the registry.</p>
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										<br /><a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/a/a6/Unitn_project_results_blh_prov1.jpg" title="Bacterial pellet confirmes &beta;-carotene production in transformed cells"><img src="https://static.igem.org/mediawiki/2015/5/5a/Unitn_project_results_blh_prov1_thumb.jpg" alt="" style="width:100%; max-width:686px; "/></a> 
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										<p>Cells transformed with this device were grown and induced for 24 hours with 5 mM arabinose. After the cells were centrifuged, we compared the two pellets to discover that also the uninduced sample showed a very bright orange pellet. The aracpBAD promoter is very tightly regulated and has a very little basal expression. By looking at the sequence of the operon, we discovered a possible internal promoter in the ctrE gene, the first gene of the pathway.</p><br />
										
										<p class="image_caption lateral-left"><span> Bacterial pellet confirmes &beta;-carotene production in transformed cells</span> A: Pellet collected from <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K731201" target="_blank">BBa_K731201</a> transformed and arabinose induced cells (control). B-C:  pellet collected from  <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604020" target="_blank">BBa_K1604020</a> (&beta;-carotene producers)  induced (B) and non induced (C). <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604020" target="_blank">BBa_K1604020</a> transformed cells produced  &beta;-carotene both in presence and absence of the arabinose induction. </p>
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										<p>Our goal was to synthetize retinal and provide it to proteorhodopsin. Our blh producing device was synthetized by Genescript<sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_2')" href="#refs_2">[2]</a></sup> with a sequence optimized for <i>E. coli</i> expression. We subcloned it into pSB1C3 and submit it to the registry (<a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604021" target="_blank">BBa_K1604021</a>). It was not possible to characterize blh in vivo by itself, due to the low solubility of &beta;-carotene and the fact that the cells would not uptake the molecule. To overcome this problem we decided to produce endogenous &beta;-carotene. Blh, under the control of a constitutive promoter J23100, was characterized both in co-transformed cells with <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604020" target="_blank">BBa_K1604020</a> (aracpBAD-ctrEBI) and in a complete device containing the complete pathway (<a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604022" target="_blank">BBa_K1604022</a>).</p>
										
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											<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/a/a4/Unitn_project_results_blh_prov2.jpg" title=""><img src="https://static.igem.org/mediawiki/2015/4/40/Unitn_project_results_blh_prov2_thumb.jpg" alt="" style="width:100%; "/></a>
											
											<p class="image_caption"><span>&beta;-carotene is absent in blh-expresing cells</span> A: Pellet collected from <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604020" target="_blank">BBa_K1604020</a> (&beta;-carotene producer) transformed cells induced with arabinose. B-C-D: Pellet collected from  BBa_K1604022 (retinal producer) transformed  cells not induced (B), induced with arabinose 5mM (C), and with both arabinose 5 mM and Fe (D);  Control cells (<a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K731030" target="_blank">BBa_K731030</a>) induced with arabinose and Fe<sup>2+</sup>(E);</p> 
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										<h4 class="header4 lateral-icon wow animated fadeInDown delay05"> <span>Where is our retinal?</span> <i class="faabig flaticon-ask3"></i></h4>
										
										<p>Happy for this result we extracted with acetone the pigments<sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_3')" href="#refs_3">[3]</a></sup> (carotenoids and retinoids) from cells expressing <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604022" target="_blank">BBa_K1604022</a> and run them on HPLC reverse phase column. &beta;-carotene was disappeared, however we did not detect any peak with the corresponding retention time of retinal.</p>
										
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											<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/5/5f/Unitn_project_results_blh_3graph.png" alt=""><img src="https://static.igem.org/mediawiki/2015/4/48/Unitn_project_results_blh_3graph_thumb.png" alt="" style="width:100%; "/></a>
											
											<p class="image_caption"><span>HPLC profile </span> Here the comparison between the HPLC profile between reference (mixture of pure &beta;-carotene and retinal), extract from <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604020" target="_blank">BBa_K1604020</a> inducted cells and <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_1604022" target="_blank">BBa_1604022</a> inducted cells.</p> 
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										<p>We also measured the absorption spectrum of the extracted pigments. We used pure retinal and &beta;-carotene for reference, which absorb at 373 nm and 456 nm respectively. Also this test confirmed the disappearance of &beta;-carotene, but did not show proof of retinal synthesis. The same behavior was observed in co-transformed cells.</p>
										
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											<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/1/1e/Unitn_project_results_blh_adsorb.png" alt=""><img src="https://static.igem.org/mediawiki/2015/d/d9/Unitn_project_results_blh_adsorb_thumb.png" alt="" style="width:100%; "/></a>
											
											<p class="image_caption"><span>Absorption spectra of cellular extracts</span> An UV-Visible assay was performed on &beta; carotenoids extracted from cells with acetone. Absorption spectrum of cells trasnfected with <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604020" target="_blank">BBa_K1604020</a>  (&beta;-carotene producers)  (yellow) resembles the spectra of a &beta;-carotene reference sample. Conversely, cells transformed with <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604022" target="_blank">BBa_K1604022</a> (red) and retinal reference do not show such analogy.</p> 
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										<p>We think that &beta;-carotene is being cleaved to form retinal (as shown by the evident loss of color, and the absence of a peak in the HPLC or UV-VIS spectrum), and is immediately taken by the cell to enter different biochemical pathways. The biosynthesis of retinal involves the formation of intermediate molecules that could also be used by <span class="bacterium"> E. coli </span> in different metabolic reactions.</p>
										 
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									<h3 class="wow animated fadeInDown">To sum up...</h3>
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										<span class="rotate-box-icon " ><i class="faa flaticon-laboratory4"></i></span> 
									</div><br />Orange is the new black</h4>  
									 <p>We confirm the functional of <span class="i_enph">ctrE</span>, <span class="i_enph">ctrB</span>, <span class="i_enph">ctrI</span>, <span class="i_enph">ctrY</span> enzymes in &beta;-carotene production.</p> 
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									</div><br />blh cleaves &beta;-carotene?</h4>  
									 <p>We observe the lack of &beta;-carotene in blh expressing cells, but were unable to characterize the presence of retinal.</p> 
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										<h3 class="wow fadeInDown">References</h3>
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										<li>Martinez, A., A. S. Bradley, J. R. Waldbauer, R. E. Summons, and E. F. Delong.<br/>
										<a href="http://www.ncbi.nlm.nih.gov/pubmed/17372221" target="_blank" class="sourcebox-link">"Proteorhodopsin Photosystem Gene Expression Enables Photophosphorylation in a Heterologous Host"</a>
										Proceedings of the National Academy of Sciences 104.13 (2007): 5590-595. Web.</li>
										
										<a class="anchor-off" name="refs_2" id="refs_2"></a> 
										<li><a href="http://www.genscript.com/" target="_blank" class="sourcebox-link">http://www.genscript.com/</a></li>
										
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										<li><a href="https://2009.igem.org/Team:Cambridge/Project/CA03" target="_blank" class="sourcebox-link">2009 Cambridge iGEM Team</a></li>  
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